Niacinamide mitigates SASP-related inflammation induced by environmental stressors in human epidermal keratinocytes and skin.

OBJECTIVE: To evaluate whether niacinamide (Nam) can mitigate production of inflammatory and senescence-related biomarkers induced by environmental stressors. METHODS: Human epidermal keratinocytes were exposed to UVB, urban dust, diesel exhaust and cigarette smoke extract and treated with Nam or vehicle control. Full thickness 3-D skin organotypic models were exposed to a combination of UVB and PM2.5 and treated with Nam or vehicle control. Quantitation of the SASP-related inflammatory mediators PGE2 , IL-6 and IL-8 was performed on cultured media. UVB-exposed keratinocytes treated with and without Nam were immunostained for the senescence biomarker Lamin B1 (LmnB1). Transcriptomics profiling of cigarette smoke extract effects on keratinocytes was performed. A double-blind, placebo-controlled clinical was conducted on 40 female panellists that were pretreated on back sites for two weeks with 5% Nam or vehicle and then exposed to 1.5 minimal erythemal dose (MED) solar-simulated radiation (SSR). Treated sites were compared with non-treated exposed sites for erythema and the skin surface IL-1αRA/IL-1α inflammatory biomarkers. RESULTS: Ultraviolet B induced synthesis of PGE2 , IL-8 and IL-6 and reduced LmnB1 levels in keratinocytes. Urban dust and diesel exhaust only stimulated synthesis of IL-8 whereas cigarette smoke extract only stimulated levels of PGE2 . In all exposures, treatment with Nam significantly mitigated synthesis of the inflammatory mediators and restored levels of UVB-reduced LmnB1. In the 3D skin equivalent model, Nam reduced IL-8 levels stimulated by a combination of topical PM2.5 and UV exposure. In a UV challenge clinical, pretreatment with 5% Nam reduced erythema and skin surface IL-1αRA/IL-1α inflammatory biomarkers that were induced by SSR. CONCLUSION: Since it is known that Nam has anti-inflammatory properties, we tested whether Nam can inhibit environmental stress-induced inflammation and senescence-associated secretory phenotype (SASP) biomarkers. We show Nam can reduce PGE2 , IL-6 and IL-8 levels induced by environmental stressors. Additionally, in vivo pretreatment with Nam can reduce UV-induced erythema and skin surface inflammatory biomarkers. These findings add to the body of evidence that Nam can mitigate the skin's inflammatory response elicited by environmental stressors. This supports Nam can potentially inhibit senescence and premature ageing and thereby maintain skin's functionality and appearance.

OBJECTIF: Évaluer si le niacinamide (Nam) peut atténuer la production de biomarqueurs inflammatoireset liés à la sénescence induits par les facteurs de stress environnementaux. MÉTHODES: Leskératinocytes épidermiques H uman ont été exposés aux UVB, à la poussière urbaine, aux gaz d'échappement diesel et à l'extrait de fumée de cigarette et traités avec nam ou contrôle de véhicule. Les modèles organotypic de peau 3D de pleine épaisseur ont été exposés à une combinaison d'UVB et de PM2.5 et traités avec nam ou commande de véhicule. La quantitation des médiateurs inflammatoires liés à la SASP PGE2 ,IL-6 et IL-8 a été réalisée sur des médias cultivés. Les kératinocytes exposés aux UVB traités avec et sans Nam étaient immunotachés pour le biomarqueur de sénescence Lamin B1 (LmnB1). Le profilage de transcriptomique des effets d'extrait de fumée de cigarette sur les kératinocytes a été exécuté. Un placebo contrôlé clinique à double insu a été menée sur 40 panélistes féminins qui ont été prétraités sur les sites arrière pendant deux semaines avec 5% Nam ou véhicule, puis exposés à 1,5 dose erythémique minimale (MED) rayonnement solaire simulé (SSR). Les sites traités ont été comparés à des sites exposés non traités pour l'érythème et la surface de la peau IL-1▫RA/IL-1▫ biomarqueurs inflammatoiress. RÉSULTATS: Synthèse induite par UVB des niveaux de PGE2, IL-8 et IL-6 et réduit de LmnB1 dans les kératinocytes. La poussière urbaine et les gaz d'échappement diesel n'ont stimulé que la synthèse de l'IL-8 alors que l'extrait de fumée de cigarette ne stimulait que les niveaux de PGE2 . Dans toutes les expositions, le traitement avec Nam a significativement atténué la synthèse des médiateurs inflammatoires et les niveaux restaurés de LmnB1 UVB-réduit. Dans le modèle équivalent de la peau 3D, Nam a réduit les niveaux d'IL-8 stimulés par une combinaison de PM combination of topical PM 2.5 topique et d'exposition aux UV. Dans un uv-défi clinique, prétraitement avec 5% Nam réduit érythème et la surface de la peau IL-1▫RA/IL-1▫ biomarqueurs inflammatoires qui ont été induits par SSR. CONCLUSION: Puisqu'il est connu que Nam a des propriétés anti-inflammatoires, nous avons testé si Nam peut inhiber l'inflammation induite par le stressenvironnementaltion et les biomarqueurs sécrétoires sécrétoires sécrétoires (SASP) associés à la sénescence. We montrent Nam peut réduire PGE2 ,IL-6, et IL-8 niveaux induits par les facteurs de stress environnementaux. En outre, le prétraitement in vivo avec Nam peut réduire l'érythème induit par les UV et les biomarqueurs inflammatoires de surface de la peau. Ces résultats ajoutent à l'oody bde la preuve que Nam peut atténuer la réponse inflammatoire de la peau provoquée par lesfacteurs de stress environnementaux. Cela soutient Nam peut potentiellement inhiber la sénescence et le vieillissement prématuré et ainsi maintenir la fonctionnalité de la peau et l'apparence.

Ligand-Independent Adenosine A2B Receptor Constitutive Activity as a Promoter of Prostate Cancer Cell Proliferation.

Aberrant ligand-independent G protein-coupled receptor constitutive activity has been implicated in the pathophysiology of a number of cancers. The adenosine A2B receptor (A2BAR) is dynamically upregulated under pathologic conditions associated with a hypoxic microenvironment, including solid tumors. This, in turn, may amplify ligand-independent A2BAR signal transduction. The contribution of A2BAR constitutive activity to disease progression is currently unknown yet of fundamental importance, as the preferred therapeutic modality for drugs designed to reduce A2BAR constitutive activity would be inverse agonism as opposed to neutral antagonism. The current study investigated A2BAR constitutive activity in a heterologous expression system and a native 22Rv1 human prostate cancer cell line exposed to hypoxic conditions (2% O2). The A2BAR inverse agonists, ZM241385 [4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol] or PSB-603 (8-(4-(4-(4-chlorophenyl)piperazide-1-sulfonyl)phenyl)-1-propylxanthine), mediated a concentration-dependent decrease in baseline cAMP levels in both cellular systems. Proliferation of multiple prostate cancer cell lines was also attenuated in the presence of PSB-603. Importantly, both the decrease in baseline cAMP accumulation and the reduction of proliferation were not influenced by the addition of adenosine deaminase, demonstrating that these effects are not dependent on stimulation of A2BARs by the endogenous agonist adenosine. Our study is the first to reveal that wild-type human A2BARs have high constitutive activity in both model and native cells. Furthermore, our findings demonstrate that this ligand-independent A2BAR constitutive activity is sufficient to promote prostate cancer cell proliferation in vitro. More broadly, A2BAR constitutive activity may have wider, currently unappreciated implications in pathologic conditions associated with a hypoxic microenvironment.

FT23, an orally active antifibrotic compound, attenuates structural and functional abnormalities in an experimental model of diabetic cardiomyopathy.

Diabetic cardiomyopathy is characterized by early diastolic dysfunction and structural changes, such as interstitial fibrosis and cardiac hypertrophy. Using the Ren-2 rat model, we sought to investigate the effect of FT23 on the structural and functional changes associated with diabetic cardiomyopathy. Heterozygous Ren-2 rats were rendered diabetic with streptozotocin by tail vein injection. Rats were then treated with FT23 (200 mg/kg per day by gavage twice daily) or vehicle from Week 8 to Week 16 after the onset of diabetes. Echocardiography was performed to assess heart function before the rats were killed and their hearts collected for histological and molecular biological assessment. The antifibrotic effect of FT23 was compared with that of tranilast in neonatal cardiac fibroblasts when stimulated with transforming growth factor (TGF)-ß (5 ng/mL) at 30, 50 and 100 umol/L. FT23 exhibited greater inhibition of TGF-ß-induced collagen production in neonatal cardiac fibroblasts, as measured by a [(3) H]-proline incorporation assay, compared with its parental compound tranilast. In the in vivo study, FT23 significantly attenuated the increased heart weight : bodyweight ratio in FT23-treated diabetic Ren-2 rats. Diastolic dysfunction, as measured by mitral valve (MV) E/A ratio and MV deceleration time, was also significantly attenuated by FT23. Picrosirius red-stained heart sections revealed that cardiac fibrosis in the diabetic rats was reduced by FT23 compared with that in vehicle-treated rats, with a concomitant reduction in collagen I immunostaining and infiltration of macrophages, as demonstrated by ED1 immunostaining. The results of the present study suggest that FT23 inhibits the activity of TGF-ß and attenuates structural and functional manifestations of diastolic dysfunction observed in a model of diabetic cardiomyopathy.

Transcription factors Krüppel-like factor 6 and peroxisome proliferator-activated receptor-{gamma} mediate high glucose-induced thioredoxin-interacting protein.

We demonstrated recently that thioredoxin-interacting protein (Txnip) and the transcription factor Krüppel-like factor 6 (KLF6) were up-regulated in both in vivo and in vitro models of diabetic nephropathy, thus promoting renal injury. Conversely, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists have been shown to be renoprotective. Hence, this study was undertaken to determine whether Txnip expression is regulated by the transcription factors KLF6 and PPAR-gamma. By using siRNAs and overexpressing constructs, the role of KLF6 and PPAR-gamma in Txnip transcriptional regulation was determined in human kidney proximal tubule cells and in streptozocin-induced diabetes mellitus in Sprague-Dawley rats, in vitro and in vivo models of diabetic nephropathy, respectively. KLF6 overexpression increased Txnip expression and promoter activity, which was inhibited by concurrent exposure to PPAR-gamma agonists. In contrast, reduced expression of KLF6 by siRNA or exposure to PPAR-gamma agonists attenuated high glucose-induced Txnip expression and promoter activity. KLF6-Txnip promoter binding was decreased in KLF6-silenced cells, whereas PPAR-gamma agonists increased PPAR-gamma-Txnip promoter binding. Indeed, silencing of KLF6 increased PPAR-gamma expression, suggesting endogenous regulation of PPAR-gamma expression by KLF6. Moreover, renal KLF6 and Txnip expression increased in rats with diabetes mellitus and was inhibited by PPAR-gamma agonist treatment; however, KLF6 expression did not change in HK-2 cells exposed to PPAR-gamma agonists. Hence, Txnip expression and promoter activity are mediated via divergent effects of KLF6 and PPAR-gamma transcriptional regulation.

α1A-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes.

The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the α1A-adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α1A-AR signalling in CHO-K1 cells co-expressing the human α1A-AR and GLUT4 (CHOα1AGLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca2+ mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α1A-AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α1-AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHOα1AGLUT4myc cells, siRNA directed against rictor but not raptor suppressed α1A-AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α1A-AR agonists. Our findings identify a novel link between the α1A-AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α1A-AR selective agonists as tools in the treatment of cardiac dysfunction.

Thioredoxin interacting protein (TXNIP) regulates tubular autophagy and mitophagy in diabetic nephropathy through the mTOR signaling pathway.

Hyperglycemia upregulates thioredoxin interacting protein (TXNIP) expression, which in turn induces ROS production, inflammatory and fibrotic responses in the diabetic kidney. Dysregulation of autophagy contributes to the development of diabetic nephropathy. However, the interaction of TXNIP with autophagy/mitophagy in diabetic nephropathy is unknown. In this study, streptozotocin-induced diabetic rats were given TXNIP DNAzyme or scrambled DNAzyme for 12 weeks respectively. Fibrotic markers, mitochondrial function and mitochondrial reactive oxygen species (mtROS) were assessed in kidneys. Tubular autophagy and mitophagy were determined in kidneys from both human and rats with diabetic nephropathy. TXNIP and autophagic signaling molecules were examined. TXNIP DNAzyme dramatically attenuated extracellular matrix deposition in the diabetic kidneys compared to the control DNAzyme. Accumulation of autophagosomes and reduced autophagic clearance were shown in tubular cells of human diabetic compared to non-diabetic kidneys, which was reversed by TXNIP DNAzyme. High glucose induced mitochondrial dysfunction and mtROS production, and inhibited mitophagy in proximal tubular cells, which was reversed by TXNIP siRNA. TXNIP inhibition suppressed diabetes-induced BNIP3 expression and activation of the mTOR signaling pathway. Collectively, hyperglycemia-induced TXNIP contributes to the dysregulation of tubular autophagy and mitophagy in diabetic nephropathy through activation of the mTOR signaling pathway.

Thioredoxin-interacting protein: a potential therapeutic target for treatment of progressive fibrosis in diabetic nephropathy.

Thioredoxin-interacting protein (TXNIP) is an endogenous inhibitor of the antioxidant thioredoxin, and a critical agent in the in vivo regulation of glucose. The well-described induction of TXNIP by high glucose may represent an important pathogenic trigger of complications arising in the diabetic environment, with sustained overexpression of TXNIP triggering the increased production of reactive oxygen species and collagen, both major contributors to the development of diabetic nephropathy (DN). To examine a possible therapeutic role for targeted TXNIP inhibition in DN, transgenic (mRen-2)27 rats were rendered diabetic with streptozotocin and then treated with 20 µM TXNIP deoxyribozyme (DNAzyme) delivered continuously over 12 weeks by an implanted osmotic mini-pump. Renal injury was measured using biochemical parameters of kidney function along with histological markers of damage. Catalytic activity of TXNIP DNAzyme was determined by TXNIP gene and peptide expression in the rat kidneys. TXNIP DNAzyme localization was demonstrated with a fluorescent-labelled TXNIP DNAzyme. A panel of markers was used to assess the extent of oxidative stress and renal fibrosis including superoxide level, nitrotyrosine staining, TGF-ß1, NLRP3 and collagen IV expression. Fluorescent-labelled TXNIP DNAzyme was localized to tubulo-epithelial cells, but was not identified in glomeruli or endothelial cells. Elevated renal cortical TXNIP gene and protein expression seen in kidneys of DN animals were significantly attenuated by TXNIP DNAzyme (p < 0.05). Downstream markers of TXNIP activity, particularly oxidative stress, inflammasome signalling, tubulo-interstitial fibrosis and collagen deposition, were also attenuated in the tubulo-interstitium of DN rats treated with TXNIP DNAzyme. Consistent with the identified site of action of the DNAzyme, the effects of the TXNIP inhibition were limited to the tubulo-interstitial compartment. This study supports the role of TXNIP as an important mediator of progressive tubulo-interstitial fibrosis in DN, and also supports the notion of TXNIP inhibition as a potential new therapeutic target for DN.

The roles of Kruppel-like factor 6 and peroxisome proliferator-activated receptor-γ in the regulation of macrophage inflammatory protein-3α at early onset of diabetes.

Macrophage inflammatory protein-3 alpha (MIP-3α) is known to be upregulated early in the development of diabetic nephropathy (DN). However, the transcriptional regulation of MIP-3α is unknown. We previously demonstrated that the transcription factors KLF6 and PPAR-γ play key roles in regulating renal fibrotic and inflammatory responses to factors inherent in diabetes mellitus. Hence we determined the role of these transcription factors in regulating MIP-3α expression. HK-2 cells and STZ-induced diabetic rats were used. siRNAs, over-expressing constructs and CHIP promoter binding assays were used to determine the role of KLF6 and PPAR-γ in MIP-3α transcriptional regulation. KLF6 overexpression increased MIP-3α which was inhibited by concurrent exposure to PPAR-γ agonists. PPAR-γ agonists attenuated high glucose-induced MIP-3α secretion. Furthermore, MIP-3α secretion was up-regulated in PPAR-γ silenced cells, suggesting both KLF6 and PPAR-γ antagonistically regulate high glucose-induced MIP-3α secretion. The CHIP promoter binding assay confirmed that PPAR-γ binds to the MIP-3α promoter and negatively regulates MIP-3α expression. PPAR-γ agonists increased the binding activity of the PPAR-γ-MIP-3α promoter. In contrast, promoter binding activity decreased in KLF6 over-expressing cells. PPAR-γ decreased in KLF6 over-expressing cells and increased in KLF6 silenced cells, while PPAR-γ siRNA had no effect on KLF6 expression, suggesting that KLF6 acted upstream of PPAR-γ in the regulation of MIP-3α. In diabetic rats, renal MIP-3α and the macrophage marker ED-1 expression increased, which was inhibited by exposure to PPAR-γ agonists. The recognition of MIP-3α as a significant pathogenic mediator in diabetic nephropathy reaffirms the increasingly recognized role of inflammation in the progression of DN. Targeting pro-inflammatory chemokine MIP-3α and its signaling pathways will provide novel strategy to treat diabetic kidney disease.